BACKGROUND: The
application of ancillary molecular testing is becoming more important
for the diagnosis and classification of disease. The use of fine-needle
aspiration (FNA) biopsy as the means of sampling tumors in conjunction
with molecular testing could be a powerful combination. FNA is
minimally invasive, cost effective, and usually demonstrates accuracy
comparable to diagnoses based on excisional biopsies. Quality control
(QC) and test validation requirements for development of molecular
tests impose a need for access to pre-existing clinical samples. Tissue
banking of excisional biopsy specimens is frequently performed at large
research institutions, but few have developed protocols for
preservation of cytologic specimens. This study aimed to evaluate
cryopreservation of FNA specimens as a method of maintaining cellular
morphology and ribonucleic acid (RNA) integrity in banked tissues.METHODS: FNA specimens
were obtained from fresh tumor resections, processed by using a
cryopreservation protocol, and stored for up to 27 weeks. Upon
retrieval, samples were made into slides for morphological evaluation,
and RNA was extracted and assessed for integrity by using the Agilent
Bioanalyzer (Agilent Technologies, Santa Clara, Calif).
RESULTS: Cryopreserved specimens showed good cell morphology and, in
many cases, yielded intact RNA. Cases showing moderate or severe RNA
degradation could generally be associated with prolonged specimen
handling or sampling of necrotic areas.CONCLUSIONS: FNA
specimens can be stored in a manner that maintains cellular morphology
and RNA integrity necessary for studies of gene expression. In addition
to addressing quality control (QC) and test validation needs, cytology
banks will be an invaluable resource for future molecular morphologic
and diagnostic research studies.Maintaining RNA integrity in a homogeneous
population of mammary epithelial cells isolated by Laser Capture
Microdissection.
Bevilacqua C, Makhzami S, Helbling JC, Defrenaix P, Martin P.
BMC Cell Biol. 2010 Dec 6;11:95.
INRA, UMR1313 Unité Génétique Animale et Biologie
Intégrative, équipe Lait, Génome &
Santé F-78350 Jouy-en-Josas, France

BACKGROUND: Laser-capture
microdissection (LCM) that enables the isolation of specific cell
populations from complex tissues under morphological control is
increasingly used for subsequent gene expression studies in cell
biology by methods such as real-time quantitative PCR (qPCR),
microarrays and most recently by RNA-sequencing. Challenges are i) to
select precisely and efficiently cells of interest and ii) to maintain
RNA integrity. The mammary gland which is a complex and heterogeneous
tissue, consists of multiple cell types, changing in relative
proportion during its development and thus hampering gene expression
profiling comparison on whole tissue between physiological stages.
During lactation, mammary epithelial cells (MEC) are predominant.
However several other cell types, including myoepithelial (MMC) and
immune cells are present, making it difficult to precisely determine
the specificity of gene expression to the cell type of origin. In this
work, an optimized reliable procedure for producing RNA from alveolar
epithelial cells isolated from frozen histological sections of
lactating goat, sheep and cow mammary glands using an infrared-laser
based Arcturus Veritas LCM (Applied Biosystems®) system has been
developed. The following steps of the microdissection workflow:
cryosectioning, staining, dehydration and harvesting of microdissected
cells have been carefully considered and designed to ensure cell
capture efficiency without compromising RNA integrity.

RESULTS: The best results were
obtained when staining 8 μm-thick sections with Cresyl violet®
(Ambion, Applied Biosystems®) and capturing microdissected cells
during less than 2 hours before RNA extraction. In addition, particular
attention was paid to animal preparation before biopsies or
slaughtering (milking) and freezing of tissue blocks which were
embedded in a cryoprotective compound before being immersed in
isopentane. The amount of RNA thus obtained from ca.150 to 250 acini
(300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity
number (RIN) was ca. 8.0 and selectivity of this LCM protocol was
demonstrated through qPCR analyses for several alveolar cell specific
genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well
as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of
MMC, lymphocytes and macrophages, respectively.

BACKGROUND: Gene
expression profiling is a highly sensitive technique which is used for
profiling tumor samples for medical prognosis. RNA quality and
degradation influence the analysis results of gene expression profiles.
The impact of this influence on the profiles and its medical impact is
not fully understood. As patient samples are very valuable for clinical
studies, it is necessary to establish criteria for the RNA quality to
be able to use these samples in later analysis.METHODS: To investigate
the effects of RNA integrity on gene expression profiling, whole genome
expression arrays were used. We used tumor biopsies from patients
diagnosed with locally advanced rectal cancer. To simulate degradation,
the isolated total RNA of all patients was subjected to heat-induced
degradation in a time-dependent manner. Expression profiling was then
performed and data were analyzed bioinformatically to assess the
differences.RESULTS: The differences
introduced by RNA degradation were largely outweighed by the biological
differences between the patients. Only a relatively small number of
probes (275 out of 41,000) show a significant effect due to
degradation. The genes that show the strongest effect due to RNA
degradation were, especially, those with short mRNAs and probe
positions near the 5' end.CONCLUSIONS: Degraded
RNA from tumor samples (RIN > 5) can still be used to perform gene
expression analysis. A much higher biological variance between patients
is observed compared to the effect that is imposed by degradation of
RNA. Nevertheless there are genes, very short ones and those with the
probe binding side close to the 5' end that should be excluded from
gene expression analysis when working with degraded RNA. These results
are limited to the Agilent 44 k microarray platform and should be
carefully interpreted when transferring to other settings.

Messenger RNA (mRNA)
profiling in post-mortem human tissue might reveal information about
gene expression at the time point of death or close to it. When working
with post-mortem human tissue, one is confronted with a natural RNA
degradation caused by several parameters which are not yet fully
understood. The aims of the present study were to analyse the influence
of impaired RNA integrity on the reliability of quantitative gene
expression data and to identify ante- and post-mortem parameters that
might lead to reduced RNA integrities in post-mortem human brain,
cardiac muscle and skeletal muscle tissues. Furthermore, this study
determined the impact of several parameters like type of tissue, age at
death, gender and body mass index (BMI), as well as duration of agony,
cause of death and post-mortem interval on the RNA integrity. The
influence of RNA integrity on the reliability of quantitative gene
expression data was analysed by generating degradation profiles for
three gene transcripts. Based on the deduced cycle of quantification
data, this study shows that reverse transcription quantitative
polymerase chain reaction (RT-qPCR) performance is affected by impaired
RNA integrity. Depending on the transcript and tissue type, a shift in
cycle threshold values of up to two cycles was observed. Determining
RNA integrity number of 136 post-mortem samples revealed significantly
different RNA qualities among the three tissue types with brain
revealing significantly lower integrities compared to skeletal and
cardiac muscle. The body mass index was found to influence RNA
integrity in skeletal muscle tissue (M. iliopsoas). Samples originating
from deceased with a BMI > 25 were of significantly lower integrity
compared to samples from normal weight donors. Correct data
normalisation was found to partly diminish the effects caused by
impaired RNA quality. Nevertheless, it can be concluded that in
post-mortem tissue with low RNA integrity numbers, the detection of
large differences in gene expression activities might still be
possible, whereas small expression differences are prone to
misinterpretation due to degradation. Thus, when working with
post-mortem samples, we recommend generating degradation profiles for
all transcripts of interest in order to reveal detection limits of
RT-qPCR assays.

BACKGROUND: There is great interest
in circulating microRNAs (miRNAs) as disease biomarkers. Translating
promising miRNAs into validated clinical tests requires the
characterization of many preanalytical and analytical parameters.

METHODS: miRNAs were extracted from
serum and plasma samples of healthy volunteers, and miRNAs known to be
present in serum and plasma (miR-15b, miR-16, miR-24, and miR-122) were
amplified by reverse-transcription quantitative PCR. Stability and the
effects of hemolysis were determined. Assay variation and its
components, including the effect of adding control miRNA, were assessed
by nested ANOVA.

RESULTS: miRNA concentrations were
higher in plasma than in serum. Processing of plasma to remove
subcellular/cellular components reduced miRNA concentrations to those
of serum. The miRNAs analyzed were stable refrigerated or frozen for up
to 72 h and were stable at room temperature for 24 h. Hemolysis
increased the apparent concentration of 3 of the miRNAs. The total
variability of replicate miRNA concentrations was <2.0-fold, with
most of the variability attributable to the extraction process and
interassay imprecision. Normalizing results to those of spiked
exogenous control miRNAs did not improve this variability.

CONCLUSIONS: Detailed validation of
the preanalytical steps affecting miRNA detection and quantification is
critical when considering the use of individual miRNAs as clinical
biomarkers. Unless these causes of imprecision are considered and
mitigated, only miRNAs that are extremely up- or downregulated will be
suitable as clinical biomarkers.

Quantitative reverse
transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate
RNA abundance. The procedure is highly sensitive and reproducible as
long as the initial RNA is intact. However, breaks in the RNA due to
chemical or enzymatic cleavage may reduce the number of RNA molecules
that contain intact amplicons. As a consequence, the number of
molecules available for amplification decreases. We determined the
relation between RNA fragmentation and threshold values (Ct values) in
subsequent QRT-PCR for four genes in an experimental model of intact
and partially hydrolyzed RNA derived from a cell line and we describe
the relation between RNA integrity, amplicon size and Ct values in this
biologically homogenous system. We demonstrate that degradation-related
shifts of Ct values can be compensated by calculating delta Ct values
between test genes and the mean values of several control genes. These
delta Ct values are less sensitive to fragmentation of the RNA and are
unaffected by varying amounts of input RNA. The feasibility of the
procedure was demonstrated by comparing Ct values from a larger panel
of genes in intact and in partially degraded RNA. We compared Ct values
from intact RNA derived from well-preserved tumor material and from
fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE)
samples of the same tumors. We demonstrate that the relative abundance
of gene expression can be based on FFPE material even when the amount
of RNA in the sample and the extent of fragmentation are not known.

Highly sensitive
techniques for transcriptome analysis, such as microarrays,
complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs),
and others currently used in functional genomics require a high RNA
quality and integrity, as well as reproducibility among extractions of
replicates from the same tissue. There are, however, few technical
papers comparing different homogenization techniques and reagents to
extract RNA from small quantities of plant tissue. We extracted RNA
from tomato seedlings with the three different commercial reagents
TRIZOL LS, TRIZOL, and TRI Reagent in combination with pulverization,
homogenization-maceration in a mortar, and homogenization with mild
vibration plus glass beads, and evaluated total RNA integrity-quality
and yield. Pulverization under liquid nitrogen combined with TRIZOL LS
as extraction reagent and homogenization-maceration in mortar with TRI
Reagent, are the procedures that rendered higher RNA yield, integrity
and quality, as well as reproducibility among independent RNA
extractions. In contrast, short mild vibration pulses (4500r.p.m. for
5s) mixed with glass beads, rendered low extraction efficiency and
caused, in most cases, partial RNA degradation.

The effect of RNA
degradation on the diagnostic utility of microRNA has not been
systematically evaluated in clinical samples. We asked if the microRNA
profile is preserved in degraded RNA samples derived from mouse and
human tissue. We selected tissue-specific microRNA candidates from
published human microarray data, and validated them using quantitative
reverse transcription polymerase chain reaction (QRTPCR) analyses on
flash-frozen, normal mouse liver, pancreas, and stomach tissue samples.
MiR-122a, miR-1, and miR-200b were identified as tissue-specific, and
the 3-microRNA-based QRTPCR could predict the tissue origin for mouse
tissue samples that were left at room temperature for 2 h with an
accuracy of 91.7%. When we applied this 3-microRNA predictor to
clinical specimens with various degree of RNA degradation, the
predictor differentiated degraded RNA samples from liver, pancreas, and
stomach with an accuracy of 90% (26/29). Expression levels of miR-122a,
miR-1, and miR-200b were modestly changed after the extended (2-4 h)
storage at room temperature, but the magnitudes of expression changes
were small compared to the expression differences between various
tissues of origin. This proof-of-principle study demonstrates that RNA
degradation due to extended storage at room temperature does not affect
the predictive power of tissue-specific microRNA QRTPCR predictor.A
robust RNA integrity-preserving staining protocol for laser capture
microdissection of endometrial cancer tissue.
Cummings M, McGinley CV, Wilkinson N, Field SL, Duffy SR, Orsi NM.
Anal Biochem. 2011 Sep 1;416(1): 123-125
Gynaeimmunology and Oncology Group, YCR and Liz Dawn Pathology and
Translational Sciences Centre, Leeds Institute of Molecular Medicine,
St. James's University Hospital, Leeds LS9 7TF, UK.

BACKGROUND: Purification
of mRNA from stored specimens is very important because results from
RT-PCR and microarray analyses are largely affected by the quality of
mRNA. Moreover, many preanalytical factors during collection,
processing, and storage may affect mRNA quality and the expression of
peripheral blood mononuclear cells (PBMC). In this study, we evaluate
the effects of RBC removal techniques and TRIzol on RNA quality in
blood samples.METHODS: We obtained
EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy
coats for comparison between protocols and also evaluated RNA quality
of clinical samples in biobank. Use of TRIzol and RBC removal (RBC
lysis or cell separation) were evaluated their effect on the quality of
mRNA from the stored blood samples.RESULTS: RNA integrity
with TRIzol was significantly better than that without TRIzol (RIN 4.5
vs. 9.2, respectively; P=0.002). The change in RIN of the PBMC
separation method was equivalent to that of the RBC lysis method. After
12months, IL6 mRNA expression from stored clinical samples in cell
separation/TRIzol was stable.CONCLUSIONS: The blood
samples frozen in TRIzol after RBC removal preserved RNA quality well.
PBMC/TRIzol preservation for storage of blood samples could be a simple
protocol for rapid, low-cost biobanking.Effects of delay in the snap freezing of
colorectal cancer tissues on the quality of DNA and RNA.
Hong SH, Baek HA, Jang KY, Chung MJ, Moon WS, Kang MJ, Lee DG, Park HS.
J Korean Soc Coloproctol. 2010 Oct;26(5): 316-323
Chonbuk National University Hospital National Biobank of Korea, Jeonju,
Korea.

PURPOSE: The success of
basic molecular research using biospecimens strongly depends on the
quality of the specimen. In this study, we evaluated the effects of
delayed freezing time on the stability of DNA and RNA in fresh frozen
tissue from patients with colorectal cancer.METHODS: Tissues were
frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal
cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A(260)/A(280))
and agarose gel electrophoresis were evaluated. In addition, the RNA
integrity number (RIN) was assayed for the analysis of the RNA
integrity.RESULTS: Regardless of
delayed freezing time, all DNA and RNA samples revealed A(260)/A(280)
ratios of more than 1.9, and all DNA samples showed a discrete,
high-molecular-weight band on agarose gel electrophoresis. The RINs
were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and
4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though
the concentration of RNA was not affected by delayed freezing, the RNA
integrity was decreased with increasing delayed freezing time.CONCLUSION: According to
the RIN results, we recommend that the collection of colorectal cancer
tissue should be done within 10 minutes for studies requiring RNA of
high quality and within 30 minutes for usual RNA studies.Improved protocol for high-quality
co-extraction of DNA and RNA from rumen digesta.
Popova M, Martin C, Morgavi DP.
Folia Microbiol (Praha). 2010 Jul;55(4): 368-372
INRA Clermont-Ferrand-Theix, 63122, Saint Gènes Champanelle,
France.

We report an improved method for total nucleic acids extraction from
rumen content samples. The method employs bead beating, and
phenol-chloroform extraction followed by saline-alcohol precipitation.
Total nucleic acids and RNA yield and purity were assessed by
spectrophotometric measurements; RNA integrity was estimated using
Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method
provided total nucleic acids and RNA extracts of good quantity and
quality. The extraction is not time consuming and it is valuable for
ecological studies of rumen microbial community structure and gene
expression.

Molecular investigations
gain increasing interest in forensic medicine. Examination of gene
expression levels at the time point of death might have the power to
become a complementing tool to the current methods for the
determination of cause and circumstances of death. This includes
pathophysiological conditions of disease and injury as well as the
duration of agony or other premortem factors. Additionally, recent
developments in forensic genetics revealed that tissue specific mRNAs
can be used to determine the type of body fluid present in a crime
scene stain. Although RNA is known to be rather instable, RNA could be
extracted in adequate quality from tissue samples collected during
medico-legal autopsy. Nevertheless, working with human postmortem
tissue means to deal with highly variable RNA integrities. This review
aims to give a brief overview of the possible advantages of postmortem
mRNA profiling and to shed further light into the limitations of this
method arising from reduced RNA integrities.

Using human postmortem
tissues for gene expression studies is particularly challenging.
Besides the problem of impaired RNA one has to face a very high degree
of biological variance within a sample set. Variations of individual
parameters like age, body mass, health, but also the cause and
circumstances of death and the postmortem interval lead to a rather
inhomogeneous collection of samples. To meet these problems it is
necessary to consider certain precautions before starting a gene
expression project. These precautions include the sample collection and
the determination of the RNA integrity, the number of replicates needed
and the methods used for reverse transcription and quantitative
polymerase chain reaction, but also the strategy for data normalisation
and data interpretation. In this article practical issues are discussed
to address some of the problems occurring in the work with postmortem
human samples obtained during medico-legal autopsy.

Background -
Laser-capture microdissection (LCM) that enables the isolation of
specific cell populations from complex tissues under morphological
control is increasingly used for subsequent gene expression studies in
cell biology by methods such as real-time quantitative PCR (qPCR),
microarrays and most recently by RNA-sequencing. Challenges are i) to
select precisely and efficiently cells of interest and ii) to maintain
RNA integrity. The mammary gland which is a complex and heterogeneous
tissue, consists of multiple cell types, changing in relative
proportion during its development and thus hampering gene expression
profiling comparison on whole tissue between physiological stages.
During lactation, mammary epithelial cells (MEC) are predominant.
However several other cell types, including myoepithelial (MMC) and
immune cells are present, making it difficult to precisely determine
the specificity of gene expression to the cell type of origin. In this
work, an optimized reliable procedure for producing RNA from alveolar
epithelial cells isolated from frozen histological sections of
lactating goat, sheep and cow mammary glands using an infrared-laser
based Arcturus Veritas LCM system has been developed. The following
steps of the microdissection workflow: cryosectioning, staining,
dehydration and harvesting of microdissected cells have been carefully
considered and designed to ensure cell capture efficiency without
compromising RNA integrity.

Results - The best
results were obtained when staining 8 um-thick sections with Cresyl
violet(Ambion) and capturing microdissected cells during less than 2
hours before RNA extraction. In addition, particular attention was paid
to animal preparation before biopsies or slaughtering (milking) and
freezing of tissue blocks which were embedded in a cryoprotective
compound before being immersed in isopentane. The amount of RNA thus
obtained from ca.150 to 250 acini (300,000 to 600,000 um^2) ranges
between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and
selectivity of this LCM protocol was demonstrated through qPCR analyses
for several alveolar cell specific genes, including LALBA
(alpha-lactalbumin) and CSN1S2 (alpha s2-casein), as well as Krt14
(cytokeratin 14), CD3e and CD68 which are specific markers of MMC,
lymphocytes and macrophages, respectively.

Conclusions - RNAs
isolated from MEC in this manner were of very good quality for
subsequent linear amplification, thus making it possible to establish a
referential gene expression profile of the healthy MEC, a useful
platform for tumor biomarker discovery.

BACKGROUND: RNA
integrity is the essential factor that determines the accuracy of mRNA
transcript measurements obtained with quantitative real-time
reverse-transcription PCR (RT-qPCR), but evidence is clearly lacking on
whether this conclusion also applies to microRNAs (miRNAs). We
evaluated this issue by comparative analysis of the dependence of miRNA
and mRNA measurements on RNA integrity in renal and prostate samples,
under both model and clinical conditions.

METHODS: Samples of
total RNA isolated from human renal tissue and Caki-2 cells, as well as
from prostate tissue and LNCaP cells, were incubated at 80 degrees C
for 5-240 min. We subsequently determined the RNA integrity number
(RIN) and used RT-qPCR to measure various miRNAs (miR-141, miR-155,
miR-200c, and miR-210 in renal samples, and miR-96, miR-130b, miR-149,
miR-205, and miR-222 in prostate samples). We similarly measured mRNAs
encoded by CDH16 (cadherin 16, KSP-cadherin), PPIA [peptidylprolyl
isomerase A (cycophilin A)], and TBP (TATA box binding protein) in
renal samples, and HIF1A [hypoxia-inducible factor 1, alpha subunit
(basic helix-loop-helix transcription factor)], HPRT1 (hypoxanthine
phosphoribosyltransferase 1), and KLK3 (kallikrein-related peptidase 3;
also known as PSA) in prostate samples. Additionally, we quantified
selected miRNAs and mRNAs in samples of RNAs with different RIN values
that we isolated from clinical samples. The effect of RIN on the miRNA
and mRNA data was assessed by linear regression analysis and group
comparison.

RESULTS: The
heat-incubation experiments of cell line and tissue RNAs showed that
RIN values had negligible or no effect on miRNA results, whereas all
mRNAs gradually decreased with decreasing RIN values. These findings
were corroborated by our findings with clinical samples.

CONCLUSIONS: Our results
suggest the stability of miRNAs to be generally robust, which makes
feasible accurate miRNA measurements with RT-qPCR, even in degraded RNA
preparations for which reliable mRNA analyses are commonly inapplicable.

In this study we
evaluate the suitability of two methods of RNA conservation in blood
samples, PAXgene and RNAlater, in combination with variable shipping
conditions for their application in multicenter studies and biobanking.
RNA yield, integrity, and purity as well as levels of selected mRNA and
microRNA species were analyzed in peripheral human blood samples
stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient
temperatures from the study centers to the central analysis laboratory.
Both examined systems were clearly appropriate for RNA stabilization in
human blood independently of the shipping conditions. The isolated RNA
is characterized by good quantity and quality and well suited for
downstream applications like quantitative RT-PCR analysis of mRNA and
microRNA. Superior yield and integrity values were received using
RNAlater. It would be reasonable to consider the pro-duction and
approval of blood collection tubes prefilled with RNAlater to
facilitate the use of this excellent RNA stabilization system in large
studies.

Polymerase chain
reaction (PCR) is recognized as a rapid, sensitive, and specific
molecular diagnostictool for the analysis of nucleic acids. However,
the sensitivity and kinetics of diagnostic PCR may be dra-matically
reduced when applied directly to biological samples, such as blood and
feces, owing to PCR-inhibitory components. As a result, pre-PCR
processing procedures have been developed to remove or reducethe
effects of PCR inhibitors. Pre-PCR processing comprises all steps prior
to the detection of PCR products,that is, sampling, sample preparation,
and deoxyribonucleic acid (DNA) amplification. The aim of
pre-PCRprocessing is to convert a complex biological sample with its
target nucleic acids/cells into PCR-amplifiable samples by combining
sample preparation and amplification conditions. Several different
pre-PCR processing strategies are used: (1) optimization of the DNA
amplification conditions by the use ofalternative DNA polymerases
and/or amplification facilitators, (2) optimization of the sample
preparationmethod, (3) optimization of the sampling method, and (4)
combinations of the different strategies. Thisreview describes
different pre-PCR processing strategies to circumvent PCR inhibition to
allow accurate andprecise DNA amplification.

The integrity of RNA is
a very critical aspect regarding downstream RNA based quantitative
analysis like RT-qPCR. Low-quality RNA can compromise the results of
such experiments. Today automated lab-on-chip capillary electrophoresis
allows rapid RNA quality and quantity determination, e.g. 2100
Bioanalyzer (Agilent Technologies) and the Experion (Bio-Rad). Both
platforms determine RNA quality using a numerical system which
represents the integrity of RNA. The Bioanalyzer offers the RIN
algorithm (RNA Integrity Number) on the Bioanalyzer 2100 and Bio-Rad
developed a new Experion software version that offers an algorithm for
calculating the RNA Quality Index (RQI).The aim of this study was to
compare both systems regarding sensitivity, reproducibility, linearity
and the influence of individual tissue extractions and different chip
runs on RNA quality and quantity determination.Overall it was confirmed
that both algorithms are very comparable and beneficial for the
determination of RNA quality for downstream applications. The Experion
showed slightly better results regarding reproducibility and absolute
sensitivity, whereas the 2100 Bioanalyzer showed a higher linearity.Quantitative
assessment of the sensitivity
of various commercial reverse transcriptases based on armored HIV RNA.
Okello JB, Rodriguez L, Poinar D, Bos K, Okwi AL, Bimenya GS,
Sewankambo NK, Henry KR, Kuch M, Poinar HN.
Department of Anthropology, McMaster Ancient DNA Centre, McMaster
University, Hamilton, Ontario, Canada. PLoS One. 2010 Nov
10;5(11):e13931.

BACKGROUND: The in-vitro
reverse transcription of RNA to its complementary DNA, catalyzed by the
enzyme reverse transcriptase, is the most fundamental step in the
quantitative RNA detection in genomic studies. As such, this step
should be as analytically sensitive, efficient and reproducible as
possible, especially when dealing with degraded or low copy RNA
samples. While there are many reverse transcriptases in the market, all
claiming to be highly sensitive, there is need for a systematic
independent comparison of their applicability in quantification of rare
RNA transcripts or low copy RNA, such as those obtained from archival
tissues.

METHODOLOGY/PRINCIPAL
FINDINGS: We performed RT-qPCR to assess the sensitivity and
reproducibility of 11 commercially available reverse transcriptases in
cDNA synthesis from low copy number RNA levels. As target RNA, we used
a serially known number of Armored HIV RNA molecules, and observed that
9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven
of which were sensitive to ∼100 copies, while only 5 were sensitive to
∼10 RNA template copies across all replicates tested. Despite their
demonstrated sensitivity, these five best performing enzymes
(Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed
considerable variation in their reproducibility as well as their
overall amplification efficiency. Accuscript and Superscript III were
the most sensitive and consistent within runs, with Accuscript and
Superscript II ranking as the most reproducible enzymes between assays.

CONCLUSIONS/SIGNIFICANCE:
We therefore recommend the use of Accuscript or Superscript III when
dealing with low copy number RNA levels, and suggest purification of
the RT reactions prior to downstream applications (eg qPCR) to augment
detection. Although the results presented in this study were based on a
viral RNA surrogate, and applied to nucleic acid lysates derived from
archival formalin-fixed paraffin embedded tissue, their relative
performance on RNA obtained from other tissue types may vary, and needs
future evaluation.Stabilizing
RNA at room temperature in RNAstable
Sharron Ohgi1, Laurent Coulon, Rolf Muller, Judy-Muller-Cohn, and
Omoshile ClementBiomatrica, Inc., 5627 Oberlin Dr, #120, San Diego, CA
92121, USA.
Biotechniques Vol. 48 (No. 6) 2010: 470

RNAstable is a novel
preservation product developed to protect RNA from degradation during
storage or shipment at ambient temperatures. The synthetic storage
medium is based on the natural principles of anhydrobiosis (meaning
“life without water”), a biological mechanism employed by some
organisms that enables their survival while dry for more than 100
years. Anhyd-robiotic organisms protect their DNA, RNA, proteins,
membranes and cellular systems for survival in a dry state and can be
revived by simple rehydration. RNAstable was designed to mimic these
unique characteristics to stabilize RNA at ambient tempera-tures for
prolonged time periods. Quantitative RT-PCR analysis demonstrates
successful amplification of RNA templates that were stored dry in
RNAstable for 29 months at room temper-ature and under accelerated
aging conditions equivalent to 12 years of room temperature storage
(elevated temperatures at 45°C). Samples were sealed inside a
moisture-barrier bag including a desiccant pack to ensure ideal storage
conditions. Rehydrated samples were used directly in reactions without
further purification and exhibited no inhibition or loss of activity.
This innovative technology prevents degradation of RNA at room
temperature and offers tremendous cost and energy savings as an
easy-to-use alternative to conventional freezer storage.

RNA folding into stable
tertiary structures is remarkably sensitive to the concentrations and
types of cations present; an understanding of the physical basis of
ion-RNA interactions is therefore a prerequisite for a quantitative
accounting of RNA stability. This article summarizes the energetic
factors that must be considered when ions interact with two different
RNA environments. "Diffuse ions" accumulate near the RNA because of the
RNA electrostatic field and remain largely hydrated. A "chelated" ion
directly contacts a specific location on the RNA surface and is held in
place by electrostatic forces. Energetic costs of ion chelation include
displacement of some of the waters of hydration by the RNA surface and
repulsion of diffuse ions. Methods are discussed for computing both the
free energy of the set of diffuse ions associated with an RNA and the
binding free energies of individual chelated ions. Such calculations
quantitatively account for the effects of Mg(2+) on RNA stability where
experimental data are available. An important conclusion is that
diffuse ions are a major factor in the stabilization of RNA tertiary
structures.

BACKGROUND: Assessment of DNA integrity and quantity remains a
bottleneck for high-throughput molecular genotyping technologies,
including next-generation sequencing. In particular, DNA extracted from
paraffin-embedded tissues, a major potential source of tumor DNA,
varies widely in quality, leading to unpredictable sequencing data. We
describe a picoliter droplet-based digital PCR method that enables
simultaneous detection of DNA integrity and the quantity of amplifiable
DNA.
METHODS: Using a multiplex assay, we detected 4 different target
lengths (78, 159, 197, and 550 bp). Assays were validated with human
genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was
validated with DNA quantities as low as 1 ng. We evaluated 12 DNA
samples extracted from paraffin-embedded lung adenocarcinoma tissues.
RESULTS: One sample contained no amplifiable DNA. The fractions of
amplifiable DNA for the 11 other samples were between 0.05% and 10.1%
for 78-bp fragments and ≤1% for longer fragments. Four samples were
chosen for enrichment and next-generation sequencing. The quality of
the sequencing data was in agreement with the results of the
DNA-integrity test. Specifically, DNA with low integrity yielded
sequencing results with lower levels of coverage and uniformity and had
higher levels of false-positive variants.
CONCLUSIONS: The development of DNA-quality assays will enable
researchers to downselect samples or process more DNA to achieve
reliable genome sequencing with the highest possible efficiency of cost
and effort, as well as minimize the waste of precious samples.Incorporation of measurement of DNA
integrity into qPCR assays.
Brisco M, Latham S, Bartley P, Morley A.
Biotechniques. 2010 Dec;49(6): 893-897.
Department of Haematology and Genetic Pathology, Flinders University
and Medical Centre, Bedford Park, South Australia, Australia.

Optimal accuracy of
quantitative PCR (qPCR) requires correction for integrity of the target
sequence. Here we combine the mathematics of the Poisson distribution
and exponential amplification to show that the frequency of lesions per
base (which prevent PCR amplification) can be derived from the slope of
the regression line between cycle threshold (Ct) and amplicon length.
We found that the amplifiable fraction (AF) of a target can be
determined from this frequency and the target length. Experimental
results from this method correlated with both the magnitude of a
damaging agent and with other measures of DNA damage. Applying the
method to a reference sequence, we determined the values for
lesions/base in control samples, as well as in the AFs of the target
sequence in qPCR samples collected from leukemic patients. The AFs used
to calculate the final qPCR result were generally >0.5, but were
<0.2 in a few samples, indicating significant degradation. We
conclude that DNA damage is not always predictable; quantifying the DNA
integrity of a sample and determining the AF of a specific qPCR target
will improve the accuracy of qPCR and aid in the interpretation of
negative results.

BACKGROUND: Molecular
diagnosis using urine is established for many sexually transmitted
diseases and is increasingly used to diagnose tumours and other
infectious diseases. Storage of urine prior to analysis, whether due to
home collection or bio-banking, is increasingly advocated yet no best
practice has emerged. Here, we examined the stability of DNA in stored
urine in two populations over 28 days.

METHODOLOGY: Urine from
40 (20 male) healthy volunteers from two populations, Italy and Zambia,
was stored at four different temperatures (RT, 4 degrees C, -20 degrees
C & -80 degrees C) with and without EDTA preservative solution.
Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human
DNA content was measured using multi-copy (ALU J) and single copy
(TLR2) targets by quantitative real-time PCR. Zambian and Italian
samples contained comparable DNA quantity at time zero. Generally, two
trends were observed during storage; no degradation, or rapid
degradation from days 0 to 7 followed by little further degradation to
28 days. The biphasic degradation was always observed in Zambia
regardless of storage conditions, but only twice in Italy.

CONCLUSION:
Site-specific differences in urine composition significantly affect the
stability of DNA during storage. Assessing the quality of stored urine
for molecular analysis, by using the type of strategy described here,
is paramount before these samples are used for molecular prognostic
monitoring, genetic analyses and disease diagnosis.

Here we describe a
method for the isolation of PCR-ready genomic DNA from various
zebrafish tissues that is based on a previously published murine
protocol. The DNA solutions are of sufficient quality to allow PCR
detection of transgenes from all commonly used zebrafish tissues. In
sperm, transgene amplification was successful even when diluted
1000-fold, allowing easy identification of transgenic founders. Given
its speed and low cost, we anticipate that the adoption of this method
will streamline DNA isolation for zebrafish research.

Background: To date PCR
detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from
Danish patients has
been unsuccessful. To establish whether non-detection was caused by
asuboptimal DNA extraction method, we tested five different DNA
extraction methods forpurification of DNA from atherosclerotic
tissue.Results: The five different DNA extraction methods were tested
on homogenate ofatherosclerotic tissue spiked with C. pneumoniae DNA or
EB, on pure C. pneumoniae DNA samplesand on whole C. pneumoniae EB.
Recovery of DNA was measured with a C. pneumoniae-specificquantitative
real-time PCR. A DNA extraction method based on DNA-binding to spin
columnswith a silica-gel membrane (DNeasy Tissue kit) showed the
highest recovery rate for the tissuesamples and pure DNA samples.
However, an automated extraction method based on magneticglass
particles (MagNA Pure) performed best on intact EB and atherosclerotic
tissue spiked withEB. The DNeasy Tissue kit and MagNA Pure methods and
the highly sensitive real-time PCR weresubsequently used on 78
atherosclerotic tissue samples from Danish patients undergoing
vascularrepair. None of the samples were positive for C. pneumoniae
DNA. The atherosclerotic sampleswere tested for inhibition by spiking
with two different, known amounts of C. pneumoniae DNA andno samples
showed inhibition.Conclusion: As a highly sensitive PCR method and an
optimised DNA extraction method wereused, non-detection in
atherosclerotic tissue from the Danish population was probably not
causedby use of inappropriate methods. However, more samples may need
to be analysed per patient tobe completely certain on this. Possible
methodological and epidemiological reasons for non-detection of C.
pneumoniae DNA in atherosclerotic tissue from the Danish population are
discussed. Further testing of DNA extraction methods is needed as this
study has shownconsiderable intra- and inter-method variation in DNA
recovery.Comparison of methods in the recovery of
nucleic acids from archival formalin-fixed paraffin-embedded autopsy
tissues.
Okello JB, Zurek J, Devault AM, Kuch M, Okwi AL, Sewankambo NK, Bimenya
GS, Poinar D, Poinar HN.
McMaster Ancient DNA Centre, Department of Anthropology, McMaster
University, Hamilton, Ontario L8S4L9, Canada
Anal Biochem. 2010 May 1;400(1): 110-117

Archival formalin-fixed paraffin-embedded (FFPE) human tissue
collections are typically in poor states of storage across the
developing world. With advances in biomolecular techniques, these
extraordinary and virtually untapped resources have become an essential
part of retrospective epidemiological studies. To successfully use such
tissues in genomic studies, scientists require high nucleic acid yields
and purity. In spite of the increasing number of FFPE tissue kits
available, few studies have analyzed their applicability in recovering
high-quality nucleic acids from archived human autopsy samples. Here we
provide a study involving 10 major extraction methods used to isolate
total nucleic acid from FFPE tissues ranging in age from 3 to 13years.
Although all 10 methods recovered quantifiable amounts of DNA, only 6
recovered quantifiable RNA, varying considerably and generally yielding
lower DNA concentrations. Overall, we show quantitatively that
TrimGen's WaxFree method and our in-house phenol-chloroform extraction
method recovered the highest yields of amplifiable DNA, with
considerable polymerase chain reaction (PCR) inhibition, whereas
Ambion's RecoverAll method recovered the most amplifiable RNA.